Q.1 Library Synthesis Core. Q.I7 Personnel: The continuing goal of the Library Synthesis Core (LSC) will be to develop and disseminate chemical methodology and synthesize chemical libraries as described in the project descriptions (section P). The LSC is subdivided between the three projects described in this proposal, though the interactions between project team members and methodology overlap will be quite extensive. Each project subgroup within the LSC will consist of one postdoctoral fellow and two graduate students. In the course of the library synthesis, the project subgroups will be responsible for methods development, library synthesis, and purity assessment. After library synthesis and purity assessment are completed, the libraries will be delivered to the Administrative and Compound Inventory Core (ACIC) for database entry and storage, and ultimately shipped to members of the Chemical Library Consortium (CLC). Within the LSC, the Assistant Director (Aaron Beeler) oversees the day-to-day management of all three projects, interacting with the postdoctoral students, graduate students, and technicians. He is also responsible for ensuring that the synthesis core accomplishes its stated goals, which include: (1) design of libraries for each project;(2) development and validation of methodologies and reaction protocols for generation and purification of chemical libraries;(3) interacting with the analytical services group for library analysis in the assessment of purity;(4) transferring the libraries to the ACIC for storage and maintenance;and (5) interacting with ACIC personnel to implement the biological and community outreach programs. The Organic Synthesis Specialists (OSS) will primarily implement library synthesis based on methodologies developed in the LSC. Information technology systems support within the Library Synthesis Core will be provided by Aruna Jain. This will mainly consist of oversight of the computers and databases used by the LSC. Dayle Acquilano, the Compound Curator, will continue to facilitate transfer of compound collections from the LSC to the ACIC. Placement of orders and other administrative duties will be provided by Paul Ferrari and Sarah Coenen. Q. 1.2 Library Synthesis: Our specific goals in library synthesis will focus on generation of discrete multimilligram quantities of compounds in pure form (>90% analytical purity). The development of a library synthesis plan will involve five stages (Figure 1). 1) Transfer and Validation Stage. In this stage, a postdoctoral fellow or graduate student responsible for methodology development will be paired with an Organic Synthesis Specialist (OSS) to adapt the methodology from the development stage to parallel synthesis. Strategies for this transition will vary depending on the chemistry involved, but will focus on evaluation of methodology scope and limitations, and utilization of supported reagents, scavengers, and purification techniques. Details of the overall workflow will be determined during this phase, including intermediate parallel work-up steps required (e.g. solid phase extraction (SPE)) and development of HPLC methods suitable for the compounds of interest. This first stage of library synthesis will rely heavily on small scale reaction blocks (48 position MiniBlock XT). This stage generally requires 4-8 weeks. 2) Synthesis of Scaffolds and Building Blocks. The synthesis of building blocks and scaffolds will be performed on scales suitable for the synthesis of 30 mg of each compound planned for library synthesis. Starting material and intermediates will be synthesized by the Organic Synthesis Specialists. The synthesis of scaffolds will be conducted in a low throughput/high output manner utilizing parallel reaction vessels capable of 50 - 200 ml reaction volumes (6 position MiniBlock XT, Syrris Atlas). In this regard, sufficient scaffold material will be prepared for library synthesis and storage of a stock amount for anticipated resynthesis efforts (2-5 g of each scaffold). Building blocks, including custom diversity reagents, will also be synthesized during this phase as required. This stage generally requires 3-7 weeks. 3) Library Rehearsal. Upon completion of scaffold and building block synthesis, we will rehearse a subset of the overall library. In order to minimize loss of custom scaffolds and building blocks, we have successfully employed analytical library rehearsal protocols involving piloting of reactions on approximately 10 jimol scale and analysis of reactions using HPLC/MS/ELSD. Library rehearsal will be conducted in 96-position reaction blocks and carried through the necessary work-up procedures. Each reaction will then be aliquoted for analysis using a Waters Acquity UPLC/UV/ELS/MS system and a Protasis MicroFlow Probe equipped with an autosampling system. Library rehearsal generally requires 1-2 weeks for completion. 4) Library Synthesis and Purification. The synthesis of preparative scale libraries will be carried out in 24 - 48 reaction arrays. Compounds will be synthesized on approximately 20-30 mg scale. Solution-phase parallel synthesis will be the main approach and will be accomplished using MiniBlock[unreadable] XT and SynthArray-24 reaction blocks. In the event that solid supported reagents or scavengers are utilized, the library synthesis will be carried out using MiniBlock systems equipped with 20 mL reaction vessels. Solid reagents such as catalysts or resins will be weighed into appropriate reaction vessels using the AutoChem FlexiWeigh system. This system is capable of weighing all types of solid materials and weights from 1 - 200 mg. All library members will be purified by the Purification Specialist using mass directed preparative HPLC (Waters FractionLynx Autopurification system equipped with a Micromass ZQ quadrapole mass spectrometer, Waters 996 diode array, and Sedere Sedex 75 ELS detectors). While many library synthesis efforts will be carried out utilizing parallel reaction blocks, the AutoChem FlexiWeigh, and digital pipettors, we will begin to incorporate microfluidic synthesis as described in Project 3. Synthesis and purification of a library generally requires 2-4 weeks. 5) Library Analysis and Compound Management. Immediately following the purification process, each compound will be evaluated for purity using analytical LC/MS/UV/ELSD (Waters Acquity System). Compounds with less than 90% purity will be subjected to a second preparative HPLC purification. If a compound is not purified to greater than 90% purity after two purification attempts, it will be removed from the library. Once all compounds have been verified for purity, they will be subjected to structural verification using the Protasis 1 minute NMR system. Upon validation of compound purity and structure, each library member will be transferred (using a Zinnser Lissy2002) to 15 x 45 mm conical bottom glass vials with septa screw tops (each vial will have a barcode and will be pre-weighed using the AutoChem FlexiWeigh) and evaporated using a GeneVac HT-4. After the weight of each sample has been determined, structural, analytical, and storage information will be entered into a secure IDBS Activity Base database. Vials will be stored in 24-position racks situated in sealed containers with Drierite. The sealed plastic containers (four 24-position racks each) will be stored in -40 [unreadable]C freezers. When compound libraries have been approved for submission to the NIH repository, samples will be dissolved in DMSO and transferred to vials provided by DPI using the Zinsser Lissy2002. New compound IDs will be entered into the database and compounds will be sent to the repository as dry samples or in DMSO as instructed. Each compound will be accompanied by electronic LC/MS. Building blocks and scaffolds will also be stored and catalogued for potential use in resynthesis efforts. Analysis of purified compounds, registration, and shipment will require two weeks. 0.1.3 Library Synthesis Core Informatics: The goal of the Synthesis Core Informatics initiative at the CMLD-BU is to develop an integrated electronic research environment software suite for all affiliated scientists. The software package will integrate synthetic procedures, compound registration, and biological data management into a single electronic data management resource. This project will be conducted in collaboration with ArtusLabs, Inc. (see letter of collaboration from Robin Smith). The project's scope will include the development of a social networking research environment that allows researchers to record their collaborative work in shared electronic laboratory notebooks. Through these shared notebooks, the software will greatly facilitate collaboration among scientific colleagues whether in the same lab or in different buildings. This tool is especially important for the CMLD-BU because key collaborations are being established with researchers at MIT. Academic or CLC collaborators will be easily accommodated in the group, and their contributions will facilitate the association of biological data to specific compounds or compound collections. The research planning software and database will maintain a large amount of information (documents, images, and spreadsheets) for compounds, reaction screens, projects, and studies. The information architecture will group related assets to individual compounds and establish connections between associated metadata, based on how people interact with the information (i.e. reaction development, catalyst, biological activity, analytical device). These metadata connections will allow researchers to track down important information more intuitively, through a variety of commonalities across contexts, rather than isolating information by study or project. The software suite will be programmed using Java and Ajax to ensure cross-platform compatibility (PC, Mac, and Linux). Individual researchers will be able to easily access their accounts and electronic laboratory notebooks via the Internet through any Web browser. Specific functionalities to be developed include an imbedded Java-based chemical drawing program, reaction planning capabilities, reaction screening using multidimensional processes, chemical library planning and construction, integration of ACD/Labs analytical software for instrumentation, and complete searching capabilities (e.g.. by keyword, substructure, reagent, scientist, etc.). Researchers will be able to associate biological data and add comments to that data as well as to compounds and procedures. This functionality is intended to facilitate and sustain collaborative research, as all researchers in the CMLD-BU and affiliated groups will have access to the electronic laboratory notebook. Through the development of this new collaborative research environment software suite, the CMLD-BU will continue to grow as a collection of scientists who operate synergistically to accomplish the goals of the CMLD program. The plan for development of the ArtusLabs software will take place from the summer of 2008 until the Spring of 2009. During the transition to the new electronic reaction planning software, the Symyx Reaction Planner will be used by researchers at the CMLD-BU. Q.1.4 Synthesis of Libraries Utilizing Current CMLD-BU Methodologies: The LSC will also be responsible for identifying methodologies developed in the CMLD-BU that have not yet been transferred to library synthesis. The following are methodologies that will be adapted to library synthesis by an Organic Synthesis Specialist. 1) Cyclopropanation of Alkynyl Isochromenes. Yamamoto and co-workers reported the synthesis of naphthyl ketones utilizing a cycloisomerization/Diels-Alder sequence starting with diyne benzaldehydes.' Inspired by these examples, we have utilized diyne benzaldehydes as potential substrates for tandem cycloisomerization processes to afford novel chemotypes. Initial studies revealed that upon treatment of diyne 1 with Cu(l), cycloisomerization occurred affording isochromene 2 (Scheme 1).2 The isochromene was readily transformed to fused cyclopropane 3 in the presence of PtCI2. pai-Acid mediated cyclopropanations of enynes have been reported,3 however, such reactions have not been reported to our knowledge utilizing isochromenes. Thus, the reaction will be further developed to incorporate a number of diversity sites which may be exploited for library synthesis (Scheme 1, inset). The fused cyclopropanes produced may also serve as substrates for further library synthesis through base-catalyzed rearrangement (Scheme 2). Enolization of 3 initiated an unanticipated rearrangement process to afford polycyclic ketone 6. In this manner, we intend to synthesize a library of 96 fused cyclopropanes of which 24 library members will be selected for synthesis of a sublibrary utilizing the base-mediated rearrangement. 2J Synthesis of indenoisoauinoline-derived libraries. An ongoing project in the CMLD-BU has focused on exploration of chemical transformations involving dihydroisoquinolines.4 We have demonstrated that in the presence of an aldehyde and scandium triflate as catalyst, dihydroisoquinoline 7 undergoes condensation to afford indenoisoquinoline 8. The relative stereochemistry of 8 was confirmed through an interesting annulation process to afford the bridged indenoisoquinoline 9. Efforts toward adaptation of this methodology to parallel synthesis will initiate with evaluation of potential diversity sites. At this point the design of a library of indenoisoquinolines will be conducted and the library synthesis completed to afford approximately 86 novel compounds. Several of the primary library members will be selected for the synthesis of a sublibrary through the bromination/annulation process. Representative library members are shown in Figure 3.